Microfluidic Processing of Leukocytes for Diagnostics Testing Summary/Abstract: New tests to quantify the numbers and functional states of key leukocyte types from blood samples offer enhanced determination and personalization of clinical diagnosis, prognosis, and treatment response. For example, labeling of >30 cell surface and intracellular target molecules can assess signaling pathway status of multiple types of normal leukocytes vs leukemia cells simultaneously, by multi- parameter flow cytometry or in the future, atomic mass spectrometry. However, current procedures to harvest blood leukocytes are expensive, time-consuming, have low cell yields, introduce cytotoxic reagents, selectively lose leukocyte subsets, and require considerable human expertise. Furthermore, multiple sequential steps are required including erythrocyte lysis, surface labeling with monoclonal antibodies (Mabs), fixation/permeablization, and intracellular labeling. Each step requires 1 or more centrifugal wash steps, resulting in cell loss and reduced research productivity. Development of an automated, reagent-free process that ensures high yield of target cells and greatly streamlines the pre-analytical workflow would be a significant benefit to research and clinical labs. Commercial goal: Market a system that combines the harvest, wash and concentration of leukocytes from blood, outperforming standard centrifugal methods. We will apply microfluidic deterministic lateral displacement (DLD), in which the paths cells take through the microfluidic system is based on size and is deterministic, i.e. absolutely determined, not subject to random processes. The key innovation of this STTR project is the use of DLD to remove leukocytes/leukemia cells from erythrocytes and also from labeling or other reagents in a continuous flow process, finally concentrating the target leukocytes/leukemia cells. This is a new use, making this application commercially novel as well as clinically beneficial. The Research Strategy leverages a collaboration among micromechanical systems experts at GPB Scientific LLC; hematopoietic cell biology and cell processing experts at University of Maryland (Civin lab); and microfluidics experts at Princeton University (Sturm-Austin lab). In Specific Aim 1, the team will create microfluidic devices to wash and concentrate leukocytes from a stream containing labeling Mabs in an automated process in a few minutes. In Specific Aim 2, the system will be applied to harvest and concentrate leukocytes labeled in whole blood, removing erythrocytes and unbound Mabs. This project focuses on leukocyte processing for flow cytometry, but the same technology could be used for many existing and new cellular and other (e.g. DNA, RNA) tests for cancer and other diseases. The value proposition is clear: by replacing erythrocyte lysis and centrifugal washing, GPB technology should increase research and clinical productivity through automated, reagent-free workflows that provide high quality cell products, enabling high value cell analyses with reproducible results. PUBLIC HEALTH RELEVANCE: Microfluidic Processing of Leukocytes for Molecular Diagnostic Testing This project is focused on achieving a breakthrough in the sample preparation of leukocytes prior to multi- parameter analysis via flow cytometry. Current methods involving centrifugation are tedious, manual processes that require expert operators and result in lost and damaged cells. Our microfluidic approach will greatly improve cell quality; streamline workflows, and lower costs. Applications include research and clinical diagnostics in cancer, infectious disease, and inflammatory disease, among other disease areas.